Showing papers in "Journal of Andrology in 1987"

Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.

Abstract: Spontaneous lipid peroxidation in washed human spermatozoa was induced by aerobic incubation at 32 C and measured by malonaldehyde production; loss of motility during the incubation was determined simultaneously. Malonaldehyde production at the point of complete loss of motility, defined as the lipoperoxidative lethal endpoint (LLE), was 0.10 +/- 0.03 nmol/10(8) cells (mean +/- SD, n = 40), and was independent of the time to complete loss of motility. Human spermatozoa produced both H2O2 and O2-. during aerobic incubation. Inhibition of superoxide dismutase in these cells with KCN showed that all the H2O2 production is due to action of the dismutase. The superoxide dismutase activity of individual human sperm samples varied between 1 and 10 U/10(8) cells, variations between samples from a single donor being nearly as great as those between different donors. The time to complete motility loss (tL) showed equal variation of 1 to 10 hours among samples. The rate of spontaneous lipid peroxidation, calculated as LLE/tL, for a given sperm sample and the superoxide dismutase activity of the same sample, determined prior to aerobic incubation, gave a good linear correlation (r = 0.97). Glutathione reductase, glutathione peroxidase, and glutathione were found to be present in human spermatozoa, but showed little variation among samples. These results suggest that superoxide dismutase plays the major role in protecting human spermatozoa against lipid peroxidation. In addition, the superoxide dismutase activity of a fresh sperm sample appears to be a good predictor of the lifetime (up to the complete loss of motility) of that particular sample, and so may prove useful in semen analysis.

Alkaline phosphatase histochemistry discriminates peritubular cells in primary rat testicular cell culture.

Abstract: Histochemical demonstration of alkaline phosphatase activity appears to be useful in identifying rat peritubular cells in primary testicular cell culture. In both frozen sections of rat testis and Mirsky's fixed, methacrylate-embedded rat testis, the reaction product localized primarily in peritubular cells, vascular endothelium and occasionally in interstitial cells, with much smaller amounts of reaction product associated with elongating spermatids in the germinal epithelium. Occasional late-stage tubules (X-XIV) showed weak reactivity in the epithelium, associated with spermatocytes or Sertoli cells. Ultrastructurally, Gomori-method reaction product was localized to peritubular cells, lymphatics, and spermatogonia in stage VII; no staining was found consistently in Sertoli cells. In isolated cell preparations enriched for Sertoli and germ cells, 1 to 8% of the cells demonstrated alkaline phosphatase activity, while greater than 50% of the cells stained positive for alkaline phosphatase activity in peritubular-enriched fractions. The histochemical demonstration of alkaline phosphatase activity can be useful for identifying peritubular cells in primary cultures of testicular cells.

Automatic analysis of human sperm motion.

Abstract: A new computerized methodology is described in which sperm movement characteristics are analyzed automatically. Video fields containing images of spermatozoa are electronically digitized, and the centroid position of each sperm head is determined. Over time, strings of centroids identify the swimming paths of individual spermatozoa. Details of path acquisition are described for human spermatozoa in semen, and include a discussion of how individual cells are identified and distinguished from each other. A diversified set of movement characteristics is computed for each spermatozoon, including two new measures of path shape based on the instantaneous turning angle. The traditional and the new measures of vigor and swimming pattern are evaluated and compared for consistency and redundancy. Analysis of data from human semen indicates that the new angular measures may be particularly useful in discriminating between spermatozoa exhibiting widely different patterns of motion.

Improvement in the quality and fertilization potential of a human sperm population using the rise technique.

Abstract: Semen from 63 individuals participating in an in vitro fertilization program was processed using a modified rise technique. Overall normal morphology was significantly improved in the rise (79.2%) as compared with the unprocessed sample (57.8%), and six of seven specific morphologic abnormalities were significantly reduced. Motility was significantly enhanced from 51.8% in the unprocessed samples to 89.1% in the rise samples. Spermatozoa recovered in the rise portion of the sample represented 5.9% of the total sample. Ultrastructural morphometry revealed that the rise was relatively free of abnormal sperm forms, acellular debris and non-sperm cellular elements as compared with the non-rise portion of the sample or a typical unprocessed sample. Volume density measurements demonstrated that only 18.1% of the volume of the non-rise sample was composed of normal spermatozoa compared with 83.4% of the volume of the rise. In a separate set of experiments utilizing 21 samples, the penetration of hamster eggs was significantly enhanced from 37.9% to 67.2% using spermatozoa from the initial washed sample and those from the rise, respectively. These data demonstrate the qualitative and quantitative improvements, as well as the increase in fertilizing potential, of the rise portion of the semen sample.

Quantification of Bovine Sperm Separation by a Swim-up Method Relationship to Sperm Motility, Integrity of Acrosomes, Sperm Migration in Polyacrylamide Gel and Fertility

Abstract: The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter. In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure. In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97). Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination. Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated. Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06). On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06). In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained. This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

Cryosurvival of human spermatozoa frozen in eight different buffer systems

Abstract: The present study was conducted to ascertain optimal cryoconditions for human spermatozoa by comparing the relative cryoprotective efficiency of eight buffer systems and assessing various cryovials and thaw rates under two freeze rates. Spermatozoa that were cryopreserved in one of four zwitterion buffers (TES-Tris-citrate-egg yolk-glycerol; TES-Tris-citrate-I) maintained higher progressive motility at 0, 1, 2, and 4 hours post-thaw as compared to cells frozen in glycerol only, citrate-egg yolk-glycerol and TES-Tris-citrate-egg yolk without glycerol (TES-Tris-citrate-III; P less than 0.01). Freezing in TES-Tris-citrate-I also resulted in spermatozoa that penetrated the furthest distance through cervical mucus and possessed the highest percent live spermatozoa when compared to other cryoprotective media. Spermatozoa were analyzed for their ability to penetrate zona-free hamster ova and no difference was found between buffers when the assay was corrected for progressive motility. After removal of seminal plasma/buffers and incubation for 2 hours in BWW, TES-Tris-citrate-II and TES-Tris-citrate-milk showed the greatest sperm longevity (P less than 0.05). Pooled semen was extended in TES-Tris-citrate-I and frozen in straws or ampoules in static N2 vapor or in pellets on dry ice. Thaw bath temperatures ranged from 0 to 37 C. Post-thaw progressive motility and cervical mucus penetration were similar in all treatment groups. In conclusion, the present results indicate the use of TES-Tris-citrate-I for cryopreservation of human spermatozoa. With this optimal cryoprotective buffer, the containers and thaw rates used have little effect on human sperm cryosurvival.

Isolation from Human Seminal Plasma of an Abundant 16-kDa Protein Originating from the Prostate, Its Identification with a 94-Residue Peptide Originally Described As β-Inhibin

Abstract: In addition to other known markers of the human prostate, it was shown that the prostatic fraction of the split ejaculate was rich in a 16-kDa protein with properties not described previously. This protein was purified from human seminal plasma using ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and gel filtration on Sephadex G-100. The purified protein showed a single prominent spot on two-dimensional gel electrophoresis. The sequence of the first 40 amino acids that could be positively identified was identical to that of a prostatic secretory protein of 94 amino acids (PSP94) previously designated as β-inhibin. Antibodies produced in rabbits against the purified protein were used to develop a radioimmunoassay. These antibodies appeared to recognize only the NH2-terminal portion of the native molecule since they did not react with a synthetic peptide composed of the 28 C-terminal residues. The radioimmunoassay showed that the concentration of the protein was 1320 ± 183 μg/ml in the seminal plasma of adult fertile men and 1134 ± 136 μg/ml in vasectomized patients. In hypertrophic and adenocarcinomatous prostates, the concentrations were 326 ± 156 and 104 ± 23 μg/ml, respectively, while values were lower than 0.060 μg/ml in the testis, epididymis, vas deferens and liver. The blood plasma concentration was 0.019 ± 0.002 μg/ml in 23 asymptomatic men 45 to 65 years old and 0.115 ± 0.036 μg/ml in eight patients with prostate cancer. These results show that PSP94 is a major protein in prostatic secretions that warrants further study in the monitoring of prostate cancer patients.

Evidence for a role of cyclooxygenase (prostaglandin synthetase) and prostaglandins in the sperm acrosome reaction and fertilization.

Abstract: Three cyclooxygenase (prostaglandin synthetase) inhibitors, indomethacin, phenylbutazone, and oxyphenbutazone, decreased fertilization in vitro when mixed with capacitated mouse spermatozoa before addition of the treated gametes to oocytes. Fertilization was inhibited whether the oocytes were intact, follicle cell-free, or both follicle cell-free and zona-free. At various concentrations of inhibitor, no effect was observed on the motility or forward progression of the spermatozoa. These cyclooxygenase inhibitors also decreased the guinea pig acrosome reaction. Inhibition of the acrosome reaction did not occur when a mixture of the prostaglandins (PGE2 or PGF2 alpha) and one of the inhibitors was added to the spermatozoa. Alone, these prostaglandins tended to enhance the rate at which the acrosome reaction took place. Lowered calcium levels reduced the occurrence of the acrosome reaction, an effect that could be reversed at least partially by the addition of PGE2. Even in the nominal absence of calcium, some acrosome reaction took place when PGE2 was present in the medium. These results support an essential role for cyclooxygenase and arachidonic acid metabolites, including prostaglandins, in the events leading to the acrosome reaction and fertilization.

Computerized videomicrographic analysis of rat sperm motility.

Abstract: Quantitative methods for the determination of the concentration, percent motility and swimming speed of human and animal spermatozoa can assist in the objective analysis of sperm and semen quality. These parameters are among the most discriminating indicators for both clinical and toxicologic assessments of reproductive function. A computerized videomicrographic analysis system to measure sperm motility characteristics in the Fischer 344 rat was characterized and compared with both manual and semi-automated videomicrographic methods (Blazak et al, 1985). The system compares favorably, both in accuracy and sensitivity, to these more conventional methods. The most variable indicator of potential reproductive function in the Fischer 344 rat is the total sperm count from the cauda epididymidis (coefficient of variation [CV] = 24%), while parameters of sperm motility vary least. These include percentage of motile cells (CV = 15%), curvilinear velocity (CV = 9%) and linearity (CV = 10%), which is a ratio of straight-line to total distance traveled. It was concluded that the computerized system may be useful for routine assessment of changes in sperm quality that may occur in the rat after exposure to toxic drugs or chemicals.

Effect of adrenal steroids on testosterone and luteinizing hormone secretion in the ram.

Abstract: The effects of adrenal steroids on testosterone and LH secretion and changes in serum cortisol levels in response to treatments were studied in the ram. Acute administration of synthetic ACTH (10 micrograms/kg BW) elevated (P less than 0.01) serum cortisol and transiently suppressed (P less than 0.05) serum testosterone and LH. Acute dexamethasone treatment suppressed (P less than 0.01) serum cortisol, testosterone and LH. Administration of vehicle had no effect (P greater than 0.10) on serum hormone levels. These data support the contention that adrenal steroids inhibit testicular endocrine function indirectly by acting at the hypothalamic or pituitary level because both ACTH and dexamethasone treatments suppressed serum LH. To differentiate between hypothalamic and pituitary sites of action, the pituitary and testicular responses to an LHRH challenge (100 micrograms) were examined in rams chronically treated with dexamethasone (5 mg i.m., twice daily for 5 days). This treatment regimen suppressed (P less than 0.01) serum cortisol levels. Compared with controls, basal testosterone levels were suppressed (P less than 0.05) in dexamethasone-treated rams; however, no effect (P greater than 0.10) on the magnitude of the testosterone response to LHRH or on either basal or LHRH-stimulated LH secretion was observed. Thus, although a direct testicular effect cannot be eliminated, these data suggest that, in the ram, adrenal steroids inhibit testicular endocrine function by action at the level of the hypothalamus.

Decrease in the Number of Human Ap and Ad Spermatogonia and in the Ap/Ad Ratio with Advancing Age New Data on the Spermatogonial Stem Cell

Abstract: The numbers of Ap and Ad spermatogonia per unit section of the testis were calculated in autopsy specimens from young adults and elderly men without testicular pathology. The number of Ap spermatogonia decreased from the 6th decade of life, whereas that of Ad spermatogonia began to decrease in the 8th decade. Although it has been reported that Ad spermatogonia are more sensitive to noxious agents than Ap spermatogonia, the involution of Ap spermatogonia precedes that of Ad spermatogonia. These findings provide new information on concepts relating to the spermatogonia precedes that of Ad spermatogonia. These findings provide new information on concepts relating to the spermatogonial stem cell in man.

Testicular Hemodynamic Changes After the Surgical Creation of a Varicocele in the Rat Intravital Microscopic Observations

Abstract: The present study investigated the effect of a surgically induced varicocele on the dynamics of testicular blood flow. The surface vasculature of the normal and the varicocele-affected testis was examined utilizing intravital epi-illumination microscopy. Application of this technique to the study of the varicocele is new. Blood flow characteristics in surface veins were studied as the surface temperature of the testis was varied. Periodic, reproducible stoppages in blood flow, determined by direct observation of the red blood cells, were seen in seven of eight sham animals at the lower temperatures. These stoppages were abolished and blood flow increased at higher temperatures; stoppages reappeared at lower temperatures. The periodic stoppages were present in only one of eight rats with a proven varicocele (P less than 0.025) at any temperature studied. This loss of blood flow regulation may be the result of a loss of testicular arteriolar tone and may explain the increase in testicular blood flow and temperature elevation observed in association with a varicocele. These findings may provide new insights into the pathophysiology of the varicocele and highlight the need to study the microvascular sequelae of this vascular abnormality.

Correlation between in vitro fertilization and human sperm density and motility.

Abstract: The conventional sperm characteristics of density (millions per milliliter) and motility, scored in a semi-subjective way, were correlated with results of an on-going in vitro fertilization and embryo transfer program. No male infertility patients were included in this study. Individual characteristics of the "successful" ejaculates are described. Sperm densities in the original ejaculate of more than 10 X 10(6) spermatozoa/ml did not significantly improve outcome (P less than 0.01). In contrast, sperm motility seemed to play the most important role, since most pregnancies (12/14) occurred using sperm samples with greater than or equal to 60% total motility (P less than 0.001). The incidence of multipronuclear fertilization is also described and discussed. These data, which were collected during 1984 in the in vitro fertilization unit of Professor R. Schoysman and coworkers (Vilvoorde, Brussels), may help to make fertilization in vitro and embryo transfer a viable method in cases of mild male subfertility, and to provide guidance in preparing some couples for the combined use of husband and donor semen if a sufficient number of oocytes are obtained.

Nitrocellulose and polyvinyl coatings prevent sperm adhesion to glass without affecting the motility of intact and demembranated human spermatozoa.

Abstract: The effectiveness of several glass coating agents in preventing sperm adherence to glass surfaces for both intact and demembranated human spermatozoa was investigated. These agents included bovine serum albumin (BSA), Sigmacote, poly glu-lys, Collodion and Formvar. The presence of at least 5% of seminal plasma in sperm suspensions prevented sperm adhesion to glass surfaces. On the other hand, 56% of washed spermatozoa resuspended in an isoosmotic buffer containing 1 mg/ml of BSA attached to glass. Collodion, Formvar and Sigmacote reduced sperm attachment to glass to 2, 5 and 7%, respectively. BSA was partially effective, with 20% sperm adherence to glass, and poly glu-lys was totally ineffective. Whereas Sigmacote and BSA coatings lacked transparency, Collodion always achieved the best light transmission. Demembranated reactivated spermatozoa were all attached to glass within 90 seconds of contact. This adhesion was prevented by Collodion and Formvar. Other agents were less effective or interfered with motility. In contrast to intact spermatozoa, demembranated spermatozoa have a very low progressiveness ratio (vector speed/track speed), a wide beating amplitude and because of their whiplash flagellar movement, their motility resembles that of hamster capacitated spermatozoa.

Reversibility of long-term effects of GnRH agonist administration on testicular histology and sperm production in the nonhuman primate.

Abstract: The present investigation evaluates the long-term effects of GnRH agonist treatment on testicular histology, sperm production and the subsequent recovery of these parameters. Four adult rhesus monkeys (M. mulatta) were treated with the GnRH agonist nafarelin (D-Nal(2)6-GnRH), released from i.m.--injected poly-D,L-lactic-co-glycolide microspheres for 20 months. Monthly injection of the GnRH agonist preparation uniformly suppressed serum levels of bioactive LH and testosterone. The size of the testis was reduced to about 30% of pretreatment. Sperm counts were suppressed to azoospermia for a total period of 53 and 77 weeks, respectively, in two monkeys and the other two animals were extremely oligozoospermic. Evaluation of testicular biopsy material after 6, 12 and 20 months of treatment revealed decreased seminiferous tubule diameter, spermatogenic disruption at the level of spermatogonia or spermatocytes, accumulation of lipid droplets and secondary lysosomes in the Sertoli cell cytoplasm, and increased thickness of the tubular wall compared with pretreatment histology. Electron microscopic examination revealed that the increased wall thickness was due to an enlargement of the inner collagen layer. No evidence of fibrosis or calcification could be obtained. Leydig cells were atrophic. Serum hormones, testis size and sperm counts returned to pretreatment values within 5 to 8, 13 to 16, and 18 weeks, respectively, after termination of treatment. Testicular histology, assessed 8 months after cessation of treatment, was indistinguishable from pretreatment. It is concluded that GnRH agonist-containing microspheres are a feasible modality for sustained administration of GnRH agonists and GnRH agonist-induced suppression of pituitary and testicular function is reversible following withdrawal of treatment. Thus, GnRH agonists may have a potential for regulation of male fertility and, presumably, also for treatment of precocious puberty.

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis.

Abstract: Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 x g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.

Morphometric Analyses of the Epididymis from Normal and Vasectomized Rats

Abstract: To provide a structural basis for region-specific biochemical activities of rat epididymal cells and to assess the morphometric effects of vasectomy, tissue and cellular morphologic parameters for each of six histologically defined regions from the epididymis of long-term sham-operated and vasectomized rats were analyzed stereologically. In sham-operated rats, tubule diameter generally increased from region 1 to region 6 (163 microns to 338 microns) while tubular wall height decreased (35 microns to 17.5 microns) as did tubular wall volume density (0.48 to 0.12). For columnar epithelial cells, the absolute cell number/region ranged from 16.5 to 5.1 X 10(6) such that region 2 greater than region 1 greater than region 5 greater than region 4 greater than region 6 greater than region 3. Based on cell volume, the largest columnar epithelial cells were found in region 3 (2607 +/- 127 microns3). The epididymal tubule wall was made up of 91% columnar epithelial cells, 5% lymphocytes, and 4% basal cells. The relatively small tubular lumen, large wall volume, and large columnar epithelial cell number in the caput (regions 1 to 3) provide the structural basis for maximizing biochemical interactions between columnar epithelial cells and spermatozoa. In contrast, the distal cauda (region 6), with its large lumen and small tubular wall volume, is structurally optimized for the function of storage, which requires minimal columnar epithelial cell interaction with spermatozoa. In vasectomized rats, mean tissue volumes for most epididymal regions were significantly greater than in sham rats. The absolute number of lymphocytes in vasectomized rats significantly increased in several regions, thus implicating them in post-vasectomy events.

Characterization of β-adrenergic receptors in dispersed rat testicular interstitial cells

Abstract: Recent studies have shown that beta-adrenergic agents stimulate steroidogenesis and cyclic AMP formation in mouse Leydig cells in culture. To obtain information about the possible presence and the characteristics of a beta-adrenergic receptor in rat testicular interstitial cells, the potent beta-adrenergic antagonist [125I]cyanopindolol (CYP) was used as ligand. Interstitial cells prepared by collagenase dispersion from rat testis were incubated with the ligand for 2 h at room temperature. [125I]cyanopindolol binds to a single class of high affinity sites at an apparent KD value of 15 pM. A number of sites of 6,600 sites/cell is measured when 0.1 microM (-) propranolol is used to determine non-specific binding. The order of potency of a series of agonists competing for [125I]cyanopindolol binding is consistent with the interaction of a beta 2-subtype receptor: zinterol greater than (-) isoproterenol greater than (-) epinephrine = salbutamol much greater than (-) norepinephrine. In addition, it was observed that the potency of a large series of specific beta 1 and beta 2 synthetic compounds for displacing [125I]cyanopindolol in rat interstitial cells is similar to the potency observed for these compounds in a typical beta 2-adrenergic tissue, the rat lung. For example, the potency of zinterol, a specific beta 2-adrenergic agonist, is 10 times higher in interstitial cells and lung than in rat heart, a typical beta 1-adrenergic tissue. Inversely, practolol, a typical beta 1-antagonist, is about 50 times more potent in rat heart than in interstitial cells and lung.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of unilateral, experimental varicocele are not mediated through the ipsilateral testis.

Abstract: Unilateral varicocele has been associated with diminished male fertility in humans and with bilateral physiologic and histologic changes in the testis of humans and laboratory animals. In particular, left varicocele in Sprague-Dawley rats results in bilateral increases in testicular temperature and blood flow. The mechanism by which unilateral varicocele can cause testicular changes is not known. The purpose of the present study was to determine whether or not the presence of either the ipsilateral or contralateral testicle is necessary for these effects of the varicocele to occur in the opposite testis. Varicoceles were created in adult, male rats by partial constriction of the left renal vein. Bilateral testicular blood flow was measured by a radiolabelled microsphere distribution technique and testicular temperature was taken with a needle probe thermometer. Right or left orchiectomies were performed on selected animals at the time of surgery to establish the unilateral left varicocele. Animals were studied 30 days after surgery. Mean testicular blood flow was significantly increased (P less than 0.01) in all animals having a left varicocele when compared with animals not having a varicocele regardless of whether a unilateral orchiectomy was performed. Likewise, the mean difference between intraabdominal temperature and intratesticular temperature (delta T) was significantly decreased in all groups of animals having varicoceles when compared with groups without varicoceles whether or not an orchiectomy had been performed. Thus, the studied bilateral effects of left-sided, experimental varicocele in the rat are not dependent upon the presence of a left testicle.

Biochemical Studies of Metalloendoprotease Activity in the Spermatozoa of Three Mammalian Species

Abstract: Ejaculated porcine and human spermatozoa, hamster spermatozoa from the cauda epididymidis, isolated hamster sperm heads and hamster cytoplasmic droplets contained activity that hydrolyzed the metalloendoprotease substrate ABZ-Ala-Gly-Leu-Ala-NBA (AAGLAN). Hamster sperm heads were isolated by treating spermatozoa with proteinase K and removing sperm tails with Dowex-50W beads. Hamster sperm activity was characterized using spermatozoa from which cytoplasmic droplets were removed by sonication and centrifugation. Porcine sperm preparations were essentially free of cytoplasmic droplets, while human sperm preparations retained somewhat more droplet material. Activity from all of these sources was inhibited by the metalloendoprotease inhibitors phosphoramidon, 1,10- phenanthroline, CBZ-D-Phe and CBZ-L-Phe but was not competitively inhibited by the metalloendoprotease substrate CBZ-Ser-Leu-amide. The AAGLAN hydrolyzing activity found in intact spermatozoa of all three species had a pH optimum of 6.2, while the optimum of the hamster sperm cytoplasmic droplet activity was 7.0. In addition, hamster sperm preparations were inhibited by ZnCl2 and dithiothreitol, but were not affected by toluene, benzamidine or chymostatin. The AAGLAN hydrolyzing activity of hamster sperm preparations was reduced, but not eliminated, by dialysis. It is concluded that spermatozoa from all three species, hamster sperm heads and hamster cytoplasmic droplets contain metalloendoprotease activity. Furthermore, metalloendoprotease activity found in hamster cytoplasmic droplets is different from that found in spermatozoa.

Pituitary-testicular function of prostatic cancer patients during treatment with a gonadotropin-releasing hormone agonist analog. I. Circulating hormone levels.

Abstract: Eight patients with advanced prostatic carcinoma (ages 59 to 78 years) were treated with a potent gonadotropin-releasing hormone (GnRH) agonist analog (buserelin, Hoechst; 600 micrograms intranasally, 3 times daily) and orchiectomized after 6 months of treatment. Endocrine responses were followed by serum hormone measurements during agonist treatment and for 3 months after orchiectomy. Six other patients (65 to 86 years) with advanced prostatic cancer had been orchiectomized as the first therapeutic measure and their blood samples were used as controls. In the GnRH agonist-treated patients, serum immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) decreased after initial stimulation by 70 to 80%, within 1 to 3 weeks (P less than 0.01). FSH partly recovered (P less than 0.05) after the first month of treatment. Serum prolactin (PRL) displayed a slight tendency to decline during buserelin treatment (P less than 0.05). Serum total and free testosterone (T) of the buserelin-treated patients decreased to the castrate range within 3 to 4 weeks after an initial 5-day increase (P less than 0.01). Serum progesterone and 17-hydroxyprogesterone (17-OHP-4) decreased to the castrate range (by 50 to 70%) in 1 week. Only minor changes were observed in sex hormone binding globulin (SHBG). Significant, acute elevations of LH, FSH, T, and 17-OHP-4 were observed only on day 1 after an injection of buserelin (500 microgram i.m.) and not when assessed between day 7 and month 6 of treatment. After 6 months of buserelin treatment, orchiectomy did not affect the serum steroids measured. After orchiectomy, immediate increases in serum LH, and somewhat later in FSH, were seen in the control patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of human sperm acrosin and proacrosin to semen parameters. II. Correlations.

Abstract: Previous results, simultaneously confirmed by others, suggest that a relationship exists between sperm acrosin levels and fertility in man. The assessment of sperm acrosin may therefore be a useful addition to the semen analysis, but only if the more standard semen parameter measurements cannot predict acrosin levels. Ejaculates from 102 men were analyzed and the relationship of the sperm acrosin system (acrosin, proacrosin, and acrosin inhibitor) to other seminal characteristics was determined. Very little correlation was observed between enzymatic and nonenzymatic parameters. Only five non-enzymatic parameters, all of which were morphologic, showed correlation coefficients of greater than or equal to 0.35 with acrosin and proacrosin, but none had an r-value above 0.48. The total acrosin and proacrosin levels were highly correlated to each other (r = 0.93). It is concluded that sperm acrosin/proacrosin levels cannot be predicted by other seminal parameters; thus, measurement of sperm acrosin/proacrosin may be clinically useful as a diagnostic parameters.

Male fertility and positive Chlamydial serology. A study of 61 fertile and 82 subfertile men.

Abstract: In this study the frequency and tiers of serum antibodies to chlamydia trachomatis in fertile and subfertile women and possible correlations between a positive serology and the values of conventional semen parameters were investigated The subfertile subjects were divided into 2 groups--those with and those without genitourinary infection No difference was noted between the proportion of seropositive patients in the fertile group (262%) as compared with those in the subfertile group who had no history of genitourinary infection (277%) The % of positive tests was significantly increased only among the subfertile group with past genitourinary infection (535%) No difference was found between the antibody tiers of the 2 groups Only the fertile group had any semen parameter namely the volume of the ejaculate that was significantly different between the seropositive and seronegative patients In conclusion there are 3 main issues: 1) the high frequency of serum antibodies to chlamydia trachomatis in the controls and the apparent absence of effects of a positive serology on the fertility; 2) the significant correlation between a past genitourinary infection and the presence of serum antibodies to chlamydia trachomatis; and 3) the need for a test to measure chlamydia in semen (authors modified)

Further characterization of a secreted epididymal glycoprotein in mice that binds to sperm tails.

Abstract: Sperm maturation antigen 4 (SMA-4) is a surface component of the mouse sperm tail. Previously, immunofluorescence studies indicated that SMA-4 may be secreted by principal cells of the distal caput epididymidis and bound to spermatozoa as they pass through that region of the duct. In the present study, detergent extracts of spermatozoa from the cauda epididymidis were subjected to polyacrylamide gel electrophoresis under reducing and denaturing conditions, transferred to nitrocellulose, and immunostained with a monoclonal antibody against SMA-4. A band of approximately 54,000 molecular weight was revealed. The band was also stained by the periodic acid-Schiff (PAS) procedure. This glycoprotein was not detected in extracts of spermatozoa from the proximal caput epididymidis or of spermatozoa from the cauda epididymidis that were preincubated for 4 hours in an in vitro fertilization environment. Blots of sperm-free fluid from the corpus and cauda epididymidis displayed an immunoreactive and PAS-positive band of about 85,000 molecular weight that was not observed in fluid from the caput epididymidis. The difference in the molecular weights of the antigen in the fluid and that in extracts of cauda spermatozoa suggests that SMA-4 may be modified chemically upon association with the sperm surface.

Appraising the instantaneous secretory rates of luteinizing hormone and testosterone in response to selective mu opiate receptor blockade in late pubertal boys.

Abstract: The pulsatile properties of gonadotropin and testosterone release were examined before and after chronic mu opiate receptor blockade with naltrexone, 50 mg every other day, in four normal boys in late puberty (ages 14 8/12 to 15 1/12 years). The nature of spontaneous secretory events was appraised for immunoactive LH and testosterone in blood withdrawn every 20 minutes for 24 hours, using a novel, discrete deconvolution algorithm to estimate apparent instantaneous secretory rates. The application of this methodology revealed that the frequency of discrete LH instantaneous secretory rates increased after mu opiate receptor blockade (P = 0.011). More strikingly, all parameters of testosterone secretory events responded significantly to mu opiate receptor blockade, including increases in mean estimated secretory rate (+ 47%, P = 0.02), testosterone pulse frequency (+ 64%, P < 0.001) and amplitude (+ 20%, P = 0.027). Correspondingly, decreases in testosterone interpulse secretory intervals (- 35%, P = 0.001), secretory pulse duration (- 19%, P = 0.042) and interpulse valley duration (- 35%, P = 0.006) also were noted. There was a prominent diurnal rhythm in testosterone secretion with maximal values in the morning and late evening, and marked reductions in the afternoon, sometimes to prepubertal levels. This variation in the testosterone secretory profile paralleled that of LH. In response to naltrexone, the FSH concentration series showed a significant increase in the mean FSH concentration (+ 18%) P = 0.003) and mean peak amplitude (+ 15%, P = 0.002). These data provide indirect evidence of functional coupling of the opiate system with the hypothalamic GnRH pulse generator. The marked increase in LH and testosterone secretory activity (as measured by the instantaneous secretory rate) after opiate receptor blockade suggests an integral interaction between the endogenous opiate system and the hypothalamic-pituitary gonadal axis during the later stages of puberty in males.

Correlation between defective motility (asthenospermia) and ATP reactivation of demembranated human spermatozoa.

Abstract: Human spermatozoa treated with 0.05% Triton X-100 to remove the cell membranes became immotile but flagellar movement was reinitiated by addition of 0.06 to 1 mM adenosine triphosphate (ATP). The percentage of spermatozoa showing flagellar movement 2 minutes after addition of 1 mM ATP from men with idiopathic asthenospermia (mean +/- SD, 34 +/- 15%), oligozoospermia (17 +/- 21%), sperm autoimmunity (17 +/- 12%), vasoepididymostomy (20 +/- 22%), or idiopathic zero motility (0%) was significantly lower than with spermatozoa from normal men (54 +/- 12%). The percentage of reactivated spermatozoa was correlated with the proportion of live cells (Eosin Y stain, r = 0.602, P less than 0.001), percentage of motile cells (r = 0.761, P less than 0.001), and motility index (r = 0.759, P less than 0.001) in the same semen samples. When expressed relative to initial sperm motility, the proportion reactivated was similar for idiopathic asthenospermia (85%) and normospermia (82%). Thus, failure to produce ATP does not appear to be a frequent cause of low sperm motility in man.

Follicle Stimulating Hormone, Luteinizing Hormone and Testicular Leydig Cell Responses to Estradiol Immunization in Ile‐de‐France Rams

Abstract: Active immunization of Ile-de-France rams against estradiol (E2) resulted in the production of E2-neutralizing antibodies and an elevation in the plasma concentrations of FSH, LH, and testosterone. The presence of E2 antibodies did not affect the testosterone metabolic clearance rate, indicating that the immunization-mediated 10-fold increase in plasma testosterone was the result of a 10-fold increase in testicular testosterone production. Testis weights, as well as nuclear and cytoplasmic volumes of individual peritubular and perivascular Leydig cells, were greater in E2-immunized rams than in albumin-immunized controls. Leydig cell numbers were not affected by treatment. The E2 antibodies were capable not only of neutralizing the inhibitory effects of endogenous E2 on gonadotropin levels in intact rams, but were able to block the effects of exogenously administered E2 on their FSH and LH secretory response to castration. It is concluded that circulating E2 in the ram is involved in pituitary-testicular endocrine homeostasis and that E2 immunoneutralization can be employed to enhance testosterone secretion in this species.

Tonic inhibition of adenylate cyclase in cultured hamster Sertoli cells.

Abstract: Sertoli cells cultured from immature hamsters respond to FSH with a dose-related increase in cAMP accumulation. Pertussis toxin acts synergistically with FSH to stimulate cAMP accumulation. This effect of pertussis toxin indicates that Sertoli cell adenylate cyclase is under tonic inhibition due to the activity of the Ni inhibitory transducer. The acetylcholine receptor antagonists atropine or tubocurarine, or the opioid antagonist naltrexone, have no effect on the FSH-induced stimulation of cAMP accumulation, suggesting that neither acetylcholine nor opioids are responsible for the inhibition of Sertoli cell cyclase. While exogenous adenosine is inhibitory, adenosine deaminase augments the ability of FSH to stimulate cAMP accumulation, but not to the level of pertussis toxin. This indicates that the Sertoli cells produce endogenous adenosine that is at least partially responsible for the tonic inhibition of adenylate cyclase. Other possibilities for the tonic inhibition of cyclase include other Sertoli cell products, germ cell products, peritubular cell products or an action of FSH itself through both stimulatory and inhibitory transducers.

Effects of experimental cryptorchidism and subsequent orchidopexy on seminiferous tubule functions in the lamb

Abstract: The reversibility of damage caused by cryptorchidism to the seminiferous tubules of the lamb was investigated at various ages. Lambs were made bilaterally cryptorchid either at birth or at 2 months of age. Then orchidopexy was performed at either 2 or 4 months of age. In permanently cryptorchid lambs, spermatogenesis stopped completely, and Sertoli cell function, as measured by FSH receptors, androgen receptors and ABP, was much reduced (-96%, -86% and -81%, respectively). Orchidopexy allowed the cryptorchid seminiferous epithelium to grow again, but the more differentiated the germ cells, the less they were capable of restoration. Even in 0- to 2- and 0- to 4-month-old temporarily cryptorchid lambs that had recovered normal Sertoli cell function, 16 to 49% of the tubules still were empty. It was concluded that cryptorchidism irreversibly damages the seminiferous tubules at a level other than the hormone receptors.